Human papilloma virus replication inhibitors

ABSTRACT

Methods for treating high risk HPV infection by inhibiting one or more phases of HPV replication cycle with antiviral compounds are disclosed. Moreover, a mechanism for the inhibition is suggested and targets for further antiviral compounds are disclosed.

PRIORITY

This application is a divisional application of U.S. application Ser.No. 15/573,362, filed on Nov. 10, 2017, which is a National Stage Entryof PCT/EP2016/060584, filed on May 11, 2016, which claims benefit ofU.S. provisional application 62/159,572, filed on May 11, 2015, thecontents of all of which are incorporated herein by reference.

SEQUENCE LISTING

This application contains sequence listing that is provided in computerreadable form that is identical to the written sequence listingprovided.

FIELD OF THE INVENTION

This invention is related generally to the area of inhibiting viralreplication. More specifically this invention is related to novelinhibitors of Human Papilloma Virus. Furthermore, the invention isrelated to identifying target molecules for further antiviral compounds.

BACKGROUND OF THE INVENTION

Human Papillomaviruses (HPVs) infect epithelial tissues of skin andmucosa. HPVs are small double-stranded DNA viruses which cause benignand malignant lesions in the epithelia. Infections are usually clearedby the immune system, however they can become persistent and eventuallymay develop into various cancers. To date, at least 205 different HPVtypes have been described, 12 of them (types 16, 18, 31, 33, 35, 39, 45,51, 52, 56, 58, 59) have been classified as Class 1A carcinogens, alsotermed as High-risk (HR) HPVs. Types 16 and 18 are most prevalent. Mostcommon cancer associated with HPVs is the cervical cancer: ˜500 000 newcases per year mostly in developing countries, and 266 000 deaths werereported in 2008. Moreover, HPV16 and HPV18, have been found to beassociated with about 70 invasive carcinomas of the uterine cervix. aswell as cancers of the oropharynx, anus. and other mucosal tissues.Low-risk HPVs on the other hand are not usually connected withmalignancy.

Current therapies to remove lesions caused by HPVs include variouscytodestructive procedures and immunomodulatory molecules, for exampleimiquimod. Imiquimod is not specific for HPV, but is used to treatvarious skin diseases, including skin cancer, melanoma, molluscumcontagiosum and others. In addition, there are three vaccines availableagainst HPVs: Gardasil (types 6, 11, 16 and 18), Gardasil 9 (types 16,18, 31, 33, 45, 52, 58, 6 and 11) and Cevarix (types 16 and 18). Themain active components in of these vaccines are the L1 capsid proteinsof HPV viruses. Moreover, these vaccines are only prophylactic and inaddition to this the availability of the vaccines is limited.

HPVs encode two main oncoproteins, E6 and E7, which modify cellularenvironment to be more suitable for HPV replication by inducing DNAsynthesis and cell transformation. E6 and E7 have crucial roles inHPV-related cancer development.

The life cycle of HPVs is strictly dependent on cellular differentiationprogram. Infection starts by virus entry into undifferentiated basalepithelial cells (possibly stem cells) through micro-wounds. Infectionis established by initiating initial amplification during which viralcopy number reaches up to few hundred copies per cell. Stablemaintenance phase is next, during this stage HPV genome replicatesapproximately once per cell cycle the viral copy number is constant.Viral gene expression is kept at very low levels. Vegetativeamplification and assembly of the virions takes place in highlydifferentiated cells. To replicate its genome, HPVs largely depend oncellular proteins. They themselves only encode two replication proteins:E1 and E2. E1 is an ATP-dependent DNA helicase, which initiatesreplication from the non-coding region of HPV genome—URR (LCR). It alsointeracts with various cellular replication proteins to facilitate viralreplication, for example Topoisomerase I, Replication Protein A (RPA)and Polymerase Alpha. For replication, E2 protein forms a complex withE1 and directs it to the replication origin. In addition, E2 is involvedin regulation of viral gene expression, and it tethers HPV genomes tomitotic chromosomes for efficient segregation.

Cellular DNA is constantly attacked by exogenous or endogenousDNA-damaging agents such as UV-wavelengths or errors in replication. Tocope with it, cells have very sophisticated ways to ensure integrity ofDNA, to avoid potential mutations and tumorigenesis: DNA damage responsenetwork (DDR). DDR network is orchestrated by different kinases (ATM,ATR and DNA-PK) and damage in DNA is repaired by two major pathways:Homologous recombination (HR) and Non-homologous end-joining (NHEJ). Inrecent years it has become clear that many viruses are not only capableof activating DDR but they also benefit from its activation. HPV genomesare replicated in distinct foci in the cell's nucleus which containcellular proteins necessary in DDR. By activating DDR, HPVs recruitreplication proteins to these foci which in turn help to efficientlyreplicate viral genomes. HPV replication machinery uses Homologousrecombination dependent replication during amplification of viral genomewhich results in accumulation of oligomerized molecules containing atleast two genomes.

Important proteins in DDR network are Tyrosyl-DNA-phosphodiesterasesTdp1 and Tdp2 as well as PARP1(poly-(ADP-ribose)-polymerase 1).Topoisomerases as part of Top1/Top2 cleaving complex (Top1/2cc) areproteins that locally relax otherwise tightly packed nucleic acids whichis required for replication and transcription. Several exogenous andendogenous DNA damaging agents, however cause entrapment of Top1/2cc-s.Entrapped cleaving complexes cause DNA breaks due to the collision ofreplication forks. Tdp1 is a key enzyme in repairing DNA damage causedby Top1 cc-s. It hydrolyzes phosphodiester bond between Top1 and DNA,thereby releasing Top1cc. PARP1 (Poly(ADP-ribose) polymerase-1) is anenzyme that binds to DNA and catalyzes addition of ADP-ribose polymers(PAR) to its target proteins. These modifications regulate cellularlocalization and biological activities of various proteins involved inDDR. Recently it has been suggested that PARP1 plays a key role inTdp1-dependent repair of Top1-induced DNA damage. Tdp1 inhibitorstogether with PARP1 and/or Topoisomerase 1 inhibitors have beenconsidered as targets in cancer therapy.

There are different model systems available for studying various stagesof HPV life cycle. Most of the work has been done in human primaryepithelial keratinocytes. However, using these cells is relatively timeconsuming and expensive, especially for high-throughput screening toidentify novel HPV inhibitors. In this disclosure a U2OS-based modelsystem with dual-luciferase system was used to measure cellgrowth/toxicity of the compounds and HPV genome replication suitable forhigh-throughput screening (PCT/EP2016/057898). This system allowsstudying all three replication stages of various HPVs. The geneexpression of HPVs in U2OS cells is almost identical to the one inkeratinocytes, making it suitable for identification of new anti-HPVdrugs. Moreover, replication mechanism and replication intermediates ofvarious HPV subtypes seem to be identical to the ones seen in differentkeratinocyte cell-lines.

Human Papillomaviruses are important pathogens responsible for greatnumber of various cancer cases worldwide. Regardless of the two existingvaccines, there is a need for antivirals against HPV infection becausevaccines are only preventive and other types of therapies have proven tobe unsuccessful. So far there are no specific HPV inhibitors available.High-throughput screening (HTS) of available chemical libraries iswidely used technique to identify new inhibitors against variouspathogens. However, by now there has not been a suitable model systemfor HT-screening of HPV-inhibitors.

This disclosure provides solutions to the above described problems.

SUMMARY OF THE INVENTION

This disclosure provides identification of several new HPV replicationinhibitors through high-throughput screening of NCI Diversity set IV andcustomized chemical libraries with IC50 ranging from 2.5-60 μM. Thesecompounds are highly specific to High-risk HPVs. At least four of thesecompounds inhibit Tdp1-PARP1-Top1cc pathway by characterizing theirsynergistic effect with Topoisomerase I inhibitor Campthotecin. Tdp1 andPARP1 are identified as essential cellular proteins necessary for HPVreplication, and by use of the novel inhibitors it is shown that theyare valid drug targets for the development of further antivirals.

Recently we have developed an HTS compatible Dual-luciferase basedsystem for measuring HPV genome replication (PCT/EP2016/057898). By useof this model system for screening NCI Diversity Set IV library whichconsists of different classes of biologically active compounds 5compounds were identified that inhibited HPV18 initial amplification inlow-micro molar range. None of the identified compounds inhibit E1 andE2 dependent URR replication. Besides initial amplification four out offive compounds successfully inhibited stable maintenance phase of theviral replication. In addition, three compounds inhibited vegetativeamplification which takes place in highly differentiated upperepithelia. These inhibitors or their analogues are therefore capable ofeliminating different stages of HPV infection.

Additional compounds are also suggested as potential inhibitors due tocertain structural similarities with the identified five compounds.

It is an object of this invention to provide compounds effective ineliminating different stages of HPV infection.

It is an object of his invention to treat human papilloma virus affectedcells or a subject (e.g., in vitro or in vivo).

It is an object of this invention to provide compounds for treatingcervical cancer.

It is an object of this invention to provide a method to treat HPVinfection by administering to the subject or contacting infected cellswith a pharmaceutical composition comprising one or more compounds asidentified and described in this disclosure.

In one object of this invention to provide a method of treating HPVaffected cells comprising contacting the cells with a compound describedherein or a mixture of the compounds.

In another object of this invention to provide a method of treating HPVaffected cells in a subject, comprising administering to a subject oneor more compounds or pharmaceutical compositions described herein.

It is an object of this invention to provide antiviral compound forinhibiting one or more replication stages of Human Papilloma Virus,wherein the compound is selected from the group consisting of:

and their analogs.

It is another object of this invention to provide an antiviral compoundagainst high risk HPV, wherein the compound inhibits initialamplification, stable maintenance and vegetative replication phases ofthe high risk HPV and the compound is selected from the group consistingof: NSC109128, NSC305831 and NSC82269.

It is yet another object of this invention to provide an antiviralcompound against high risk HPV, wherein the compound inhibits initialamplification and stable maintenance of high risk HPV, and the compoundis NSC88915.

Yet another object of this invention is to provide an antiviral compoundagainst high risk HPV, wherein the compound inhibits initialamplification phase of high risk HPV and the compound is NSC109128,NSC305831, NSC82269, NSC 88915 or NSC 9782.

It is an object of this invention to provide an antiviral compoundagainst HPV 16HPV 18, HPV31, HPV33, HPV45.

Still another object of this invention is to provide an antiviralcompound to inhibit one or more replication phases of Human PapillomaVirus, wherein the compound inhibits release of Topoisomerase I cleavingcomplex Top1cc from a high risk HPV DNA.

Another object of this invention is to provide an antiviral compound toinhibit one or more replication phases of HPV, wherein the compoundinhibits the release by inhibiting Tpd1 or PARP1.

It is yet another object of this invention to provide a method to treathigh risk HPV-infection, said method comprising administration of orcontacting infected cells with a compound capable of inhibiting one ormore replication phases of Human Papilloma Virus, wherein the compoundinhibits release of Topoisomerase I cleaving complex Top1cc from HPVDNA.

Still further object of this invention is to provide a method to treathigh risk HPV-infection, said method comprising administration of orcontacting infected cells with a compound capable of inhibiting one ormore replication phases of Human Papilloma Virus, wherein the compoundinhibits Tpd1 or PARP 1.

Yet another object of the invention is to provide a method to treat highrisk HPV infection said method comprising administration of orcontacting infected cell with a compound selected from the groupconsisting of:

and their analogs.

It is a further object of this invention to provide a method to inhibitone or more replication phases of high risk HPV in vitro or in vivo,said method comprising a step of contacting the infected cell with acompound inhibiting release of Topoisomerase I cleaving complex Top1ccfrom HPV DNA.

It is still another object of this invention to provide a method toinhibit one or more replication phases of high risk HPV in vitro or invivo, said method comprising a step of contacting the infected cell witha compound inhibiting Tpd1 or PARP 1.

Another object of this invention is to provide a method to inhibit oneor more replication phases of high risk HPV in vitro or in vivo, saidmethod comprising a step of contacting the infected cell with a compoundselected from the group consisting of:

and their analogs.

A further object of this invention is to provide a medicament fortreating high risk HPV infection, said medicament comprising one or moreof compounds inhibiting release of Topoisomerase I cleaving complexTop1cc from HPV DNA.

Another object of the invention is to provide a medicament for treatinghigh risk HPV infection, said medicament comprising one or more ofcompounds inhibiting Tpd1 or PARP.

Still another object of this invention is to provide a medicament fortreating high risk HPV infection, said medicament comprising one or moreof compounds selected from the group consisting of:

and their analogs.

Yet another object of this invention is to provide a method to identifypotential antiviral compounds for inhibition of high risk HPV genomereplication, said method comprising detection of compounds capable ofinhibiting Tdp1 and PARP 1.

Still another object of this invention is to provide potentialinhibitors of HPV infection or potential inhibitors of one or more HPVreplication phases, said inhibitors being selected from the groupconsisting of:

and their analogs.

Another object of the invention is to provide medicament for treatingHPV infections, said medicament comprising one or more of the compoundsselected from the group consisting of:

and their analogs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Schematic 2D-structures of the five HR-HPV replication inhibitorsidentified during the HT-screen of NCI Diversity Set IV.

FIG. 2 Effect of the identified five compounds on HPV18 initialamplification. A: U2OS cells were transfected with HPV18 wt minicircleand grown in the presence of the compounds with indicated concentrationsfor 5 days. Genomic DNA was extracted, linearized, and digested withDpnI. HPV genome replication signals were detected using Southern Blotanalyses and quantified with phosphoimager and expressed relative tovehicle control (DMSO). Approximate IC50 values are shown for eachcompound. Error-bars represent standard deviation from at least threeindependent experiment. B: U2OS cells were transfected with theexpression vectors of HPV18 E1 and E2 proteins together with HPV18 URR(origin) minicircle plasmid. The cells were grown in the presence of thecompounds with indicated concentrations for 24 or 48 hours. Genomic DNAwas extracted, linearized and digested with DpnI. HPV URR replicationsignals were detected by Southern Blot analyses. DMSO serves as vehiclecontrol and M 18URR serves as size marker.

FIG. 3 Effect of compounds on stable maintenance and vegetativeamplification of HPV18. A: U2OS #1.13 cells were grown in subconfluentconditions in the presence of the identified five compounds for 7 days.Genomic DNA was extracted, linearized and HPV replication signal wasdetected by Southern Blot and B: quantified by phosphoimager. C: U2OS#1.13 were seeded and grown for 5 days without splitting. On the 5^(th)day indicated concentrations of compounds were added to the media andthe cells were grown for additional 7 days without splitting. On the12^(th) day genomic DNA was extracted, linearized and HPV replicationsignal was detected by Southern Blot and D: quantified by phosphoimager.Error-bars represent standard deviations from at least two independentexperiments.

FIG. 4 Involvement of Tdp1 and PARP1 in HPV18 initial amplification. A:U2OS cells were transfected with HPV18 wt minicircle and sh_Tdp1plasmid. Genomic DNA was extracted 3 and 4 days after the transfection,linearized and digested with DpnI. HPV replication signal was detectedwith Southern Blot analyses and B: quantified with phosphoimager. C:Western Blot analyses showing downregulation of Tdp1 protein by shRNA.D: U2OS cells were transfected with HPV18 wt minicircle and sh_PARP1plasmid. HPV replication signal was detected with Southern Blot analysesand E: quantified with phosphoimager. F: Western Blot analyses showingdownregulation of PARP1 protein by shRNA. G: U2OS cells were transfectedwith HPV18 wt minicircle and grown for 3 and 4 days in the presence ofdifferent concentrations of PARP1 inhibitor ABT-888. HPV replicationsignal was detected with Southern Blot analyses and H: quantified withphosphoimager. Error-bars represent standard deviations from at leastthree independent experiments.

FIG. 5 Synergistic effect between Camptothecin (CPT) and the identifiedfive compounds. U2OS-EBNA1 cells were transfected with HPV18 wt and oriPplasmids and grown in the presence of indicated concentrations of thecompounds alone or together with 2 nM CPT for 5 days. Genomic DNA wasextracted, linearized and digested with DpnI. Both HPV18 and oriPreplication signals were detected with Southern Blot and quantified byphosphoimager. Compounds 109128, 88915, 9782 and 305831 show clearsynergistic inhibition together with CPT (panels C-J), whereas compound82269 does not (panels A and B). Error-bars represent standarddeviations from at least three independent experiments.

FIG. 6 Effect of the identified five compounds on different types ofHPVs. U2OS cells were transfected with 3 μg of HPV 5, 2 μg of HPV11 and5 μg of HPV16 minicircles and grown in the presence of indicatedconcentrations of compounds for 5 days. Genomic DNA was extracted,linearized and digested with DpnI. HPV replication signal was quantifiedusing qPCR. A: Effect on HPV5. B: Effect on HPV11. C: Effect on HPV16Error-bars represent standard deviations from two independentexperiments. D, E and F: U2OS cells were transfected with 2 μg HPV33minicircle, 5 μg of HPV45 minicircle and 2 μg of religated HPV31 genomeand grown in the presence of indicated concentrations of compounds for 5days. Episomal DNA was extracted using HIRT DNA extraction method,linearized and digested with DpnI. HPV replication signal detected bySouthern Blot analyses. Black arrow indicates the replicated HPV signal.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used throughout this disclosure, the following terms, unlessotherwise indicated, shall be understood to have the following meanings.If a definition is missing, the conventional definition as known to oneskilled in the art controls. If a definition provided herein conflictsor is different from a definition provided in any cited publication, thedefinition provided herein controls.

As used herein, the terms “including”, “containing”, and “comprising”are used in their open, non-limiting sense.

As used herein, the singular forms “a”, “an”, and “the” include pluralreferents unless the context clearly dictates otherwise.

As used herein, the term “subject” encompasses mammals and non-mammals.Examples of mammals include, but are not limited to, any member of theMammalian class: humans; non-human primates such as chimpanzees, andother apes and monkey species; farm animals such as cattle, horses,sheep, goats, swine; domestic animals such as rabbits, dogs, and cats;and laboratory animals including rodents, such as rats, mice and guineapigs, and the like. Examples of non-mammals include, but are not limitedto, birds, fish and the like. In one embodiment of the presentinvention, the mammal is a human.

The term “inhibitor” refers to a molecule such as a compound, a drug, anenzyme activator, or a hormone that blocks or otherwise interferes witha particular biologic activity.

The term “HPV replication inhibitor” means a compound capable ofpartially or fully to inhibit or block or interfere with one or morephases of HPV replication cycle.

The terms “effective amount” or “therapeutically effective amount” referto a sufficient amount of the agent to provide the desired biologicalresult. That result can be reduction and/or alleviation of the signs,symptoms, or causes of a disease or medical condition, or any otherdesired alteration of a biological system. For example, an “effectiveamount” for therapeutic use is the amount of a compound, or of acomposition comprising the compound, that is required to provide aclinically relevant change in a disease state, symptom, or medicalcondition. An appropriate “effective” amount in any individual case maybe determined by one of ordinary skill in the art using routineexperimentation. Thus, the expression “effective amount” generallyrefers to the quantity for which the active substance has atherapeutically desired effect.

As used herein, the terms “treat” or “treatment” encompasses both“preventive” and “curative” treatment. “Preventive” treatment is meantto indicate a postponement of development of a disease, a symptom of adisease, or medical condition, suppressing symptoms that may appear, orreducing the risk of developing or recurrence of a disease or symptom.“Curative” treatment includes reducing the severity of or suppressingthe worsening of an existing disease, symptom, or condition. Thus,treatment includes ameliorating or preventing the worsening of existingdisease symptoms, preventing additional symptoms from occurring,ameliorating or preventing the underlying metabolic causes of symptoms,inhibiting the disorder or disease, e.g., arresting the development ofthe disorder or disease, relieving the disorder or disease, causingregression of the disorder or disease, relieving a condition caused bythe disease or disorder, or stopping the symptoms of the disease ordisorder.

This invention also relates to pharmaceutically acceptable prodrugs ofthe identified compounds, and treatment methods employing suchpharmaceutically acceptable prodrugs. The term “prodrug” means aprecursor of a designated compound that, following administration to asubject, yields the compound in vivo via a chemical or physiologicalprocess such as solvolysis or enzymatic cleavage, or under physiologicalconditions (e.g., a prodrug on being brought to physiological pH isconverted to the compound). A “pharmaceutically acceptable prodrug” is aprodrug that is non-toxic, biologically tolerable, and otherwisesuitable for formulation and/or administration to the subject.Illustrative procedures for the selection and preparation of suitableprodrug derivatives are described, for example, in “Design of Prodrugs”,ed. H. Bundgaard, Elsevier, 1985. Examples of prodrugs includepharmaceutically acceptable esters of the compounds of the invention,which are also considered to be part of the invention.

Initial replication or amplification or transient replication oramplification refers to HPV DNA replication at establishment of theinfection.

Stable maintenance or latent maintenance refers to the latent stage ofviral replication cycle where viral DNA is stably maintained at analmost constant copy number in dividing host cells.

Vegetative amplificational replication or late amplificationalreplication refers to exponential viral DNA amplification in highlydifferentiated keratinocytes in the upper parts of the epithelium.

Human Papillomaviruses are important pathogens responsible for greatnumber of various cancer cases worldwide. Regardless of the twovaccines, there is still need for antivirals against HPV infectionbecause vaccines are only preventive and other types of therapies haveproven to be unsuccessful. So far there are no specific HPV inhibitorsavailable. High-throughput screening (HTS) of available chemicallibraries is widely used technique to identify new inhibitors againstvarious pathogens. However, HTS requires suitable model systems whichallow rapidly and accurately measure the effect of chemical compounds ontarget(s). Recently we have developed an HTS compatible Dual-luciferasebased system for measuring HPV genome replication (PCT/EP2016/057898).Here we used this model system for screening NCI Diversity Set IVlibrary which consists of different classes of compounds that have shownsome kind of biological activity. We identified 5 compounds (FIG. 1)that inhibited HPV18 initial amplification in low-micromolar range (FIG.2, panel A). Several studies regarding HPV replication have been carriedout by measuring E1 and E2 dependent replication of HPV URR (containsthe origin or replication) plasmid. Even HTS model system for measuringURR replication has been developed. The compounds identified here do notinhibit E1 and E2 dependent URR replication (FIG. 2, panel B),indicating that different cellular proteins could be used to facilitateHPV genome replication and/or that the replication mechanism of viralgenome differs from the URR plasmid replication. Besides initialamplification four out of five compounds successfully inhibited stablemaintenance phase of the viral replication (FIG. 3, panel A). Inaddition three compounds inhibited vegetative amplification which takesplace in highly differentiated upper epithelia (FIG. 3, panel B). Theseinhibitors or their analogues are therefore capable of eliminatingdifferent stages of HPV infection.

It has become clear in the recent years that HPVs activate DNA damageresponse network during their replication to “invite” cellularreplication and DNA repair proteins to its replication foci. Moreover,it has been shown that HPV uses Homologous recombination to efficientlyreplicate its genome. The exact mechanism and all the necessary cellularpartners are however, not known yet. Tdp1 releases entrapped Top1cc fromDNA and thereby preventing replication/transcription fork collision.Tdp1 is not absolutely necessary protein during normal replication asTop1 cleaving complexes get trapped due to certain types of DNA damage.Here we show that Tdp1 together with its regulator protein PARP1 areimportant cellular partners in HPV18 replication (FIG. 4) thus makingthose proteins good targets for developing HPV inhibitors. Camptothecin(CPT) is a Topoisomerase I inhibitor, it stabilizes Top1cc-DNA complex.In this disclosure it is shown that four of the five identifiedcompounds show synergistic inhibition of HPV18 initial amplificationwith CPT suggesting that they inhibit Tdp1or have some other targetrelated to releasing Top1cc complexes from DNA (FIG. 5). These resultssuggest that at some point during HPV replication Top1cc complexes gettrapped on the viral genome. Tdp1 seems to be then activated probably byPARP1 and recruited on HPV DNA where it releases those complexesallowing replication to continue. When Tdp1 is inhibited, abnormalreplication intermediates emerge, and HPV replication cannot becompleted. None of the compounds inhibited HPV11 or HPV5 replication(FIG. 6), which indicates that Top1cc is not be entrapped on those HPVgenomes and Tdp1 is therefore not necessary or not a limiting factor forlow-risk or cutaneous HPV replication. It is possible that high-risk HPVE1 or some other proteins interacts with Tdp1 and brings it to thereplication sites but low-risk or cutaneous HPV proteins do not.

During this work we used HPV HT-screening model system which wedeveloped to screen for compounds capable of inhibiting HPV genomereplication. We have identified five compounds: three of them inhibitall HPV replication stages, one of them inhibits initial amplificationand stable maintenance and one only the initial amplification. Inaddition, we show here for the first time that during HPV18 replication,probably due to DNA damage, Topoisomerase I cleaving complexes gettrapped on viral genome. To continue normal replication Tdp1 togetherwith PARP1 are necessary for releasing Top1cc from HPV genome.Inhibition of the release of Top1cc from HPV DNA serves as promisingtarget for antiviral development.

The present invention provides compounds for treating HPV infectedcells. The invention further provides a method of treating HPV infectedcells comprising contacting the cells with an effective amount of anantiviral compound of this the invention. A subject infected with HPVmay be treated by a method, which comprises administering to the subjectan effective amount of one or more compounds of this invention. Thecompound may be administered in the form of a pharmaceutical compositioncomprising one or more of the compounds and a pharmaceuticallyacceptable carrier.

One aspect of the invention is pharmaceutically acceptable compositionscomprising one or more antiviral compounds identified in thisdisclosure. The pharmaceutical compositions may include carriers,adjuvants or vehicle and alternatively one or more additional activeagents.

The pharmaceutical compositions, according to the method of the presentinvention, may be administered by using any amount and any route ofadministration effective for treating or lessening the severity of anHPV infection or disease.

The exact amount required will vary from subject to subject, dependingon the species, age, sex, weight, diet, medical condition and generalcondition of the subject, the severity of the infection, the particularagent, its mode of administration, and the like.

Administration of the compounds may be a single daily dose, multiple,spaced doses throughout the day, a single dose every other day, a singledose every several days or any other appropriate regimens. The dosagecan be determined routinely by using standard methods known in the art.

The pharmaceutically acceptable compositions of this invention can beadministered to humans and other animals orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, or drops), buccally, as an oral or nasal spray, orthe like, depending on the severity of the infection being treated.

Injectable preparations may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents.

Compositions for rectal or vaginal administration can be suppositorieswhich comprise one or more compounds identified in this disclosure withsuitable non-irritating excipients or carriers.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Oral formulations may also be syrups and elixirs.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis mixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Additionally, the present invention contemplates the use of transdermalpatches, which have the added advantage of providing controlled deliveryof a compound to the body. Such dosage forms are prepared by dissolvingor dispensing the compound in the proper medium. Absorption enhancerscan also be used to increase the flux of the compound across the skin.The rate can be controlled by either providing a rate controllingmembrane or by dispersing the compound in a polymer matrix or gel.

More than one compound of the invention may be administered separately,simultaneously, or sequentially to infected cells, to tissue containingthe infected cells, or to infected subjects.

It will also be appreciated that the compounds and pharmaceuticallyacceptable compositions of the present invention can be employed incombination therapies, that is, the compounds and pharmaceuticallyacceptable compositions can be administered concurrently with, prior to,or subsequent to, one or more other desired therapeutics or medicalprocedures.

The invention is now described by means of non-limiting examples.

Methods Used in the Experiments

Cell lines and transfection. U2OS (ATCC no: HTB-96), U2OS-EBNA1(Icosagen Cell Factory OÜ), U20S-GFP-Fluc #10.15 (DMSZ deposit numberACC3258) were grown in Iscove's modified Dulbecco's medium (IMDM)supplemented with 10% fetal calf serum (FCS). All U2OS cell-lines weretransfected by electroporation (220V; 975 μF) with a Bio-Rad Gene PulserX-cell supplied with a capacitance extender (Bio-Rad Laboratories).

Plasmids. The parental plasmids pMC-HPV18 for HPV18 wt minicircleproduction was constructed by adding BglII restriction site (used forcloning into pMC.BESBX vector [54]) into HPV18 genome between nt. 7473and nt. 7474 (herein, the numbering of the HPV18 genome is according tothe NCBI Reference Sequence NC_001357.1) which have been shown noalterations in the gene expression and replication. The parental plasmidpMC-HPV18-RlucE2 (DMSZ deposit number DSM 29865) was constructed basedon pMC-HPV18. The parental plasmid pMC-HPV5 was constructed by usingXmaJI restriction site in the ORF of L2 protein. The parental plasmidpMC-HPV11 was constructed by using BamHI restriction site in the HPV11genome. The parental plasmid pMC-HPV33 was constructed by using Eco81Irestriction site in the HPV33 genome. The parental plasmid pMC-HPV45 wasconstructed by using AscI restriction site in the HPV45 genome. TheHPV31 genome sequence cloned in pBR322 vector was obtained from theInternational Human Papillomavirus (HPV) Reference Center. The parentalplasmid pMC-HPV18URR contains 206 nucleotides from the end of L1 ORF,full-length HPV18 URR and 14 nucleotides from the beginning of E6 ORF.This fragment was cloned into the BglI site in pMC.BESBX vector. ThepMC.BESBX vector contains bacterial origin of replication and kanamycinresistance gene necessary for propagation in bacterial cells andadditional elements for minicircle production. Minicircle HPV genomeswere produced in E. coli strain ZYCY10P3S2T as described in [54]. ThepMC-HPV containing E. Coli strain ZYCY10P3S2T was grown in TerrificBroth media (Life Technologies) at 37° C. for 16-18 h, after which equalamount of LB media containing 0.02% L-arabinose and 20 mM NaOH was addedand culture was grown at 32° C. for 8 hours to induce recombination andproduction of supercoiled minicircles. Finally, the HPV genomes werepurified from E. coli with the QIAfilter Plasmid kit (Qiagen). Theresulting minicircle HPV genomes have less than 100 bp of non-HPVsequence. For generation of HPV31 genome for transfection, HPV31 plasmidwas digested with EcoRI, resolved in agarose gel, HPV31 sequence waspurified from gel and religated. Expression vectors for HPV18 E1 and E2are described in [36]. Ebstein Barr Virus oriP plasmid p994 was a kindgift from B. Sugden, described in [55]. ShRNA expression was under thecontrol of RNA polymerase III promoter U6.

Tdp1 shRNA sequence: (SEQ ID NO: 1)GCACGATCTCTCTGAAACAAACTCGAGTTTGTTTCAGAGAGATCGT PARP1 shRNA sequence:(SEQ ID NO: 2) GGACTCGCTCCGGATGGCCTTCAAGAGAGGCCATCCGGAGCGAGTCC

High-throughput screen for identification of HPV18 inhibitors. TheU2OS-GFP-Fluc #10.15 were transfected with 2 ug of HPV18-Rluc-E2minicircle and the cells were seeded onto 100 mm plates. On the nextday, the cells were detached and seeded onto 96-well plates (5000 cellsper well). 48 hours after the transfection screened compounds were addedto the media in 5 uM and 1 uM concentrations. The cells were grown forthree days and both Firefly luciferase (shows cellular viability) andRenilla luciferase (shows HPV copy number) were measured using Dual-Gloluciferase assay system (Promega) according to manufacturer's protocolwith the GloMAX-96 luminometer (Promega). The results were blotted on aXY-scatter diagram and HPV-specific hits were chosen.

Chemicals. The Diversity set IV and additionally compounds NSC9782, NSC88915, NSC 82269, NSC 109128 and NSC305831 were obtained from DrugSynthesis and Chemistry Branch, Developmental Therapeutics Program,Division of Cancer Treatment and Diagnosis, National Cancer InstituteUSA. Camptothecin (CPT) (sc-200871) and ABT-888 (sc-202901) werepurchased from Santa Cruz Biotechnology.

Replication assay. The U2OS or U2OS-EBNA1 cells were transfected with 2ug of HPV18 minicircle genome and lug of oriP plasmid (only in case ofU2OS-EBNA1 cells). All the compounds were added to the media immediatelyafter the transfection and media was changed on the third day.Replication signals were detected with Southern Blot method. Briefly,genomic DNA was extracted 3 and 4 days after the transfection withsh_RNAs or with the use of ABT-888 and 5 days after for analyses of theeffects of HPV inhibitors w/wo CPT. DNA was digested with BglI and DpnIfor analyses of HPV18 replication and with BstXI, ScaI and DpnI foranalyses of HPV18 and oriP replication simultaneously. 3 ug of DNA wasresolved in agarose gel, transferred to a nylon membrane (MembraneSolutions LLC) and hybridized with specific radioactively labeledprobes. Signals were detected and quantified with Typhoon Trio+phosphoimager (GE Healthcare) and exposed on an X-ray film (AGFA).

HPV copy number quantitation. Quantitative real-time PCR (qPCR) was usedto evaluate the effect the compounds had on HPV11 and HPV5 replication.U2OS cells were transfected with 2 μg of the HPV11 or HPV5 minicircle,compounds were added to the media immediately after the transfection andagain on the 3^(rd) day when the media was changed. Genomic DNA wasextracted 5 days after the transfection, the samples were linearizedwith HindIII (for HPV11) and SacI (for HPV5) and digested with DpnI tofragment input DNA. For each qPCR reaction, 3 ng of DNA was used. Thereactions were performed with EvaGreen qPCR Mix Rox (Solis BioDyne)according to the manufacturer's protocol using 7900 HT Fast Real-TimePCR System (Applied Biosystems). Oligonucleotides (300 nM of each perreaction):

HPV11: (SEQ ID NO: 3) 5′-AGCATGCAGACACATCAGGAATATTAG-3′ and(SEQ ID NO: 4) 5′-GTGCCGATTGGGTGGTTGCAGGATTTG-3′; HPV5: (SEQ ID NO: 5)5′-GGTTGCAGGAACTGTGAGGT-3′ and (SEQ ID NO: 6)5′-TCCGCGACAGTCGGGGCACAGG-3′.

The Ct_(rDNA) was detected as a normalization standard from theribosomal DNA gene sequence in the U2OS genome with the followingoligonucleotides (300 nM of each):

(SEQ ID NO: 7) 5′-GCGGCGTTATTCCCATGACC-3′ and (SEQ ID NO: 8)5′-GGTGCCCTTCCGTCAATTCC-3′.

The relative value C_(N), which reflects the average viral genome copynumber per cell, was calculated from the data withΔCt=Ct_(HPV)−Ct_(rDNA) and C_(N)=2^(−ΔCt).

Western Blot. The cells were lysed with Laemmli buffer (4% SDS, 20%glycerol, 120 mM Tris-Cl (pH 6.8), and 200 mM DTT) and boiled for 10minutes at 100° C. Samples were resolved in SDS-PAGE gel and transferredto PVDF membrane Immobilon-P (Millipore). Tdp1 and PARP1 were detectedwith their specific antibodies from Santa Cruz Biotechnology: sc-365674and sc-56197 respectively. Tubulin (used as loading control) wasdetected using Sigma Aldrich antibody T9026. Anti-mouse peroxidaseconjugated secondary antibody (LabAS) and Amersham ECL Western BlottingDetection Kit (GE Healthcare) were used for visualization. Signals wereexposed on an X-Ray film (AGFA).

Five Novel HPV Inhibitors Identified in High-Throughput Screening of NCIDiversity Set IV.

NCI Diversity set IV was screened as described in Example 1 with thepreviously characterized HPV model system (U.S. provisional application62/145,243 Tdp1 shRNA sequence:GCACGATCTCTCTGAAACAAACTCGAGTTTGTTTCAGAGAGATCGT (SEQ ID NO:1)

PARP1 shRNA sequence: (SEQ ID NO: 2)GGACTCGCTCCGGATGGCCTTCAAGAGAGGCCATCCGGAGCGAGTCC

for compounds inhibiting initial amplification of HPV18 genome in U2OScells. HT-screening was conducted with all the compounds in this libraryin 5 uM and 1 uM concentrations and it gave us 80 hits (approximately 5%of analyzed compounds). After validation of the hits on HPV18 wt genome,we found five compounds inhibiting initial amplification of HPV18 inU2OS cells (FIG. 1). To further characterize the compounds, we performedreplication assay described in Example 1. Relative inhibition to vehiclecontrol DMSO is shown on FIG. 2, panel A. All five compounds: NSC 9782,NSC82269, NSC 88915, NSC 109128 and NSC 305831, showconcentration-dependent inhibition of HPV18 initial amplification, IC50ranging from 2.5 to 60 uM.

We were curious if these identified compounds could inhibit E1 and E2dependent replication of the plasmid containing HPV origin ofreplication—URR. We transfected U2OS cells with 25 ng of E1 and E2expression vectors together with 500 ng of HPV18 URR minicircle plasmidand added various concentrations of compounds as indicated on FIG. 2,panel B. We detected HPV18 URR replication by Southern blot analyses andnone of the compounds inhibited URR replication compared to the vehiclecontrol DMSO (FIG. 2, panel B) indicating that different cellularproteins are used to facilitate HPV genome replication and/orreplication machinery of viral genome differs from the URR plasmidreplication. Another possibility is the difference in the levels of E1and E2 proteins: the expression level during HPV genome replication isconsiderably lower than the level in URR replication assay.

Effect of compounds on stable maintenance and vegetative amplification.

Our U2OScell-based system is suitable for studying all three replicationstages of various HPV subtypes. Stable maintenance phase could bemonitored by maintaining HPV-positive cells in subconfluent conditions.We used HPV18 positive monoclonal U2OS cell-line #1.13 with viral copynumber ˜2000. We grew the #1.13 cells in the presence of variousconcentration of compounds in subconfluent conditions (cells were splitevery three days) for 7 days. HPV18 replication signal was detected bySouthern blot analyses and quantified with phosphoimager. About 50% ofthe signal was lost in case of compound 82269 compared to the DMSOcontrol (FIG. 3, panel A, compare lanes 4-6 with lane 15 and panel B).Compounds 88915, 109128 and 305831 showed even higher inhibitoryeffect—about 70% of the signal was lost during the 7-day period (FIG. 3,panel A, compare lanes 7-14 with lane 15 and panel B). In case ofcompound 9782, no significant reduction in signal could be detected(FIG. 3, panel A, lanes 1-3 and panel B). Thus four of the fiveidentified compounds effectively blocked the stable maintenance of ahigh risk type HPV.

If #1.13 is grown in confluent conditions for at least 5 days,vegetative amplification of HPV genome is turned on. We seeded equalamounts of cells onto plates and grew them for 5 days without splitting.On the 5^(th) day various concentrations of compound were added and thecells were grown for additional 7 days (media was changed every two daysthroughout the experiment). HPV replication signal was detected bySouthern Blot analyses and quantified with phosphoimager. As can be seenon FIG. 3, panel C lanes 16-18, about 5-7-fold increase in the viralcopy number occurs reminiscent of vegetative amplification. Compounds109128 and 305831 almost completely abolished amplification in higherconcentrations (FIG. 3, panel C, lanes 10-15, panel D). Compound 82269also showed clear concentration-dependent inhibition of vegetativeamplification (FIG. 3, panel C, lanes 4-6 and panel B). However,compounds 9782 and 88915 did not have any effect (FIG. 3, panel C, lanes1-3 and 7-9, panel D).

In conclusion, compounds 109128, 305831 and 82269 clearly inhibit bothstable maintenance and vegetative amplification of HPV18, 88915 onlyinhibits stable maintenance phase and 9782 does not inhibit any of thelater replication stages.

Tdp1 and PARP1 are Involved in HPV18 Initial Amplification.

Two of the identified compounds, 88915 and 305831 are known to inhibitTdp. To examine if Tdp1 is necessary for HPV18 initial amplification, wetransfected U2OS cells with HPV18 genome together with variousconcentrations of shRNA_Tdp1 plasmid. Empty shRNA plasmid was used asmock control. Firstly, Tdp1 expression was evaluated and FIG. 4, panel Cshows clearly that Tdp1 expression is decreased significantly throughthe use of shRNA_Tdp1. Next genomic DNA was extracted 3 and 4 days afterthe transfection and replication assay was performed. FIG. 4, panels A(compare lanes 1 and 2 with 3-8) and B show clear decrease in HPV18replication when Tdp1 expression is downregulated.

It was recently shown that PARP1 is activating Tdp1 and recruiting it tosites of DNA damage. To examine if PARP1 is also involved in HPV18replication, we transfected U2OS with HPV18 genome together withshRNA_PARP1 plasmid or empty shRNA vector. FIG. 4, panel F show thatPARP1 expression is decreased due to shRNA_PARP1 expression. Genomic DNAwas next extracted 3 and 4 days after the transfection and replicationassay was performed to evaluate HPV18 initial amplification. FIG. 4,panels D (compare lanes 1 and 2 with 3-6) and E indeed show thatdownregulation of PARP1 inhibits HPV18 replication significantly.

PARP1 involvement in HPV18 replication was also evaluated using knownPARP1 inhibitor ABT-888 (veliparib). U2OS cells were transfected withHPV18 genome and grown in the presence of various concentrations ofABT-888 for 3 and 4 days, DMSO serves as vehicle control. Replicationassay was performed and FIG. 4, panels G (compare lanes 1 and 2 with3-10) and H show that HPV18 replication is decreased through the use ofPARP1 inhibitor ABT-888.

In conclusion the results demonstrate that both Tdp1 and PARP1 areimportant cellular partners in HPV18 replication.

Synergistic effect of Campthotecin (CPT) and HPV18 replicationinhibitors. CPT is a Topoisomerase I inhibitor that stabilizes entrappedTop1cc complexes to DNA. Since Tdp1 is responsible for cleavingentrapped Top1cc complexes from DNA, synergistic effect between CPT andTdp1 inhibitors occurs. In the experiments shown in FIG. 5 we used U2OScells constitutively expressing Epstein Barr virus (EBV) EBNA1protein—U2OS-EBNA1. These cells allow to monitor HPV and EBNA1 supportedoriP plasmid replication simultaneously. During HPV initialamplification, viral genome is replicated several times during cellcycle, however EBNA1-dependent replication occurs once per cell cycledue to cellular control mechanisms, which suggest HPV and EBV usecompletely different replication mechanisms. U2OS-EBNA1 cells werecotransfected with HPV18 genome and oriP plasmid and the cells weregrown in the presence of various concentrations of the identified HPVinhibitors alone or together with 2 nM of CPT for 5 days, DMSO was usedas vehicle control. Replication assay was performed, signals werequantified with phosphoimager and results in FIG. 5 show that all of theHPV inhibitors are highly specific because none of them inhibited EBNA1dependent replication of oriP plasmid. 2 nM CPT alone did not inhibitHPV18 initial amplification. In case of compounds 109128 (FIG. 5, panelsC and D), 88915 (panels E and F), 9782 (panels G and H) and 305831(panels I and J), clear concentration dependent synergistic effecttogether with CPT occurs as HPV replication is more efficientlyinhibited (compare lanes 1-7 with 8-12 in all experiments). Compound82269 (FIG. 5, panels A, compare lanes 1-7 with 8-12 and B) however,showed no synergistic effect together with CPT.

Compounds are not Universal HPV Inhibitors

To examine if the compounds identified here inhibit other HPV subtypesas well, U2OS cells were transfected with LR-HPV type 11 and cutaneousHPV type 5 genomes and grown in the presence of various concentrationsof compounds for 5 days, media was changed on the 3^(rd) day. DMSO wasadded as vehicle control. Genomic DNA was extracted, HPV DNA waslinearized and digested with DpnI. HPV replication signals werequantified with qPCR analyses as described in the materials and methodssection. Result on FIG. 6 show that none of the compounds inhibit HPV5(FIG. 6, panel A) or HPV 11 (FIG. 6, panel B) initial amplification.However, compounds are active against HPV16 (FIG. 6, panel C), HPV33(FIG. 6, panel D) and HPV31 FIG. 6, panel E). Accordingly, it seems thatthe compounds inhibit high risk HPV replication, including HPV 16, HPV18, HPV31 and HPV33, but not the low risk and cutaneous HPV types.

Compounds Inhibit the Replication of HR-HPVs through the HPV ReplicationProteins E1 or E2.

Besides oncoproteins E6 and E7, HPVs differ from replication proteins E1and E2 as well. To determine if the compounds identified here inhibitthe replication of HR but not LR or cutaneous HPVs through E1 or E2,HPV18E1E2 mutant genome (deficient in replication due to mutations inthe ORFs of E1 and E2) was replicated by E1 and E2 proteins originatingfrom HPV11. HPV18E1E2 mutant genome alone was used as control. U2OScells were co-transfected with HPV18E1E2 mutant and HPV11 wt mini circlegenome and the cells were grown in the presence of compound 305831 for 5days, media was changed on the 3^(rd) day. DMSO was added as vehiclecontrol. Genomic DNA was extracted, HPV DNA was digested with AgeI, SdaIand DpnI. HPV replication was detected by Southern Blot analyses.Results on FIG. 7 show, that HPV18E1E2 mutant genome is indeedreplication-deficient when transfected alone (lane 7). DMSO control onFIG. 7, lane 6 shows that HPV11 wt genome successfully complements theE1/E2 deficient HPV18 genome. In case of samples treated with thecompound 305831, there is no inhibition of HPV18 replication which iscarried out by the E1 and E2 proteins originating from HPV11 genome(FIG. 7, compare lanes 1-6 with 7). This data suggests that thecompounds target either HR-HPV E1 or E2 protein or their interactionwith cellular proteins. The activity of LR-HPV E1 and E2 proteins ortheir interactome seem to be different compared to HR-HPV replicationproteins.

Further Potential Compounds for Inhibiting HPV Replication

Based on the structure of the identified five compounds that areeffective in the inhibiting one or more phases of the HPV replication,we have identified the following three structures having certainsimilarities with the identified five compounds for potential compoundsfor use in inhibiting HPV replication.

According to preliminary experiments, these compounds seem to be capableof inhibiting the initial amplification phase of HPV 18.

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What is claimed is:
 1. A method to reduce or decrease viral replicationin high risk HPV-infection, said method comprising administration of, orcontacting infected cells with a compound capable of inhibiting one ormore replication phases of Human Papilloma Virus, wherein the compoundinhibits release of Topoisomerase I cleaving complex Top1cc from HPVDNA, wherein the compound is selected from the group consisting of:


2. The method of claim 1, wherein the release of Topoisomerase Icleaving complex Top1cc from HPV DNA is inhibited by inhibiting Tpd1 orPARP
 1. 3. The method of claim claim 1, wherein the HPV is HPV 16,HPV18, HPV31, HPV33 or HPV45.
 4. A method to inhibit one or morereplication phases of high risk HPV in vitro or in vivo, said methodcomprising a step of contacting the infected cell with a compoundinhibiting release of Topoisomerase I cleaving complex Top1cc from HPVDNA, wherein the compound is selected from the group consisting of:


5. The method of claim 4, wherein the release of Topoisomerase Icleaving complex Top1cc from HPV DNA is inhibited by inhibiting Tpd1 orPARP
 1. 6. The method of claim 4, wherein the HPV is HPV 16, HPV 18,HPV31, HPV33 or HPV45.
 7. A method to reduce or decrease viralreplication in high risk HPV infection, the method comprising a step ofadministration of, or contacting infected cells with any one of thecompounds:


8. The method of claim 7, wherein the HPV is HPV18.